The present invention relates to the field of peptide chemistry. More particularly, the invention relates to the preparation and use of peptides containing amino acid sequences substantially similar to the corresponding sequences of the kringle 5 region of mammalian plasminogen, pharmaceutical compositions containing the peptides, antibodies specific for the kringle 5 receptor, means for kringle 5 detection and measurement, cytotoxic agents linked to kringle 5 proteins and treatment of diseases which arise from or are exacerbated by angiogenesis.
Angiogenesis, the process by which new blood vessels are formed, is essential for normal body activities including reproduction, development and wound repair. Although the process is not completely understood, it is believed to involve a complex interplay of molecules which regulate the growth of endothelial cells (the primary cells of capillary blood vessels). Under normal conditions, these molecules appear to maintain the microvasculature in a quiescent state (i.e. one of no capillary growth) for prolonged periods which may last for as long as weeks or, in some cases, decades. When necessary (such as during wound repair), these same cells can undergo rapid proliferation and turnover within a 5 day period (Folkman, J. and Shing, Y., The Journal of Biological Chemistry, 267(16), 10931-10934, and Folkman, J. and Klagsbrun, M., Science, 235, 442-447 (1987).
Although angiogenesis is a highly regulated process under normal conditions, many diseases (characterized as angiogenic diseases) are driven by persistent unregulated angiogenesis. Otherwise stated, unregulated angiogenesis may either cause a particular disease directly or exascerbate an existing pathological condition. For example, ocular neovascularization has been implicated as the most common cause of blindness and dominates approximately 20 eye diseases. In certain existing conditions, such as arthritis, newly formed capillary blood vessels invade the joints and destroy cartilage. In diabetes, new capillaries formed in the retina invade the vitreous, bleed, and cause blindness. Growth and metastasis of solid tumors are also dependent on angiogenesis (Folkman, J., Cancer Research, 46, 467-473 (1986), Folkman, J., Journal of the National Cancer Institute, 82, 4-6 (1989). It has been shown, for example, that tumors which enlarge to greater than 2 mm must obtain their own blood supply and do so by inducing the growth of new capillary blood vessels. Once these new blood vessels become embedded in the tumor, they provide a means for tumor cells to enter the circulation and metastasize to distant sites such as liver, lung or bone (Weidner, N., et al., The New England Journal of Medicine, 324(1): 1-8 (1991)).
To date, several naturally occurring angiogenic factors have been described and characterized (Fidler, J. I. and Ellis, L. M., Cell, 79: 185-189 (1994)). Recently, O""Reilly, et al. have isolated and purified a 38 kilodalton (kDa) protein from serum and urine of tumor-bearing mice that inhibits endothelial cell proliferation (O""Reilly, M. et al., Cell, 79: 315-328 (1994) and International Application WO 95/29242, published Nov. 2, 1995). Microsequence analysis of this endothelial inhibitor showed 98% sequence homology to an internal fragment of murine plasminogen. Angiostatin, as the murine inhibitory fragment was named, was a peptide which included the first four kringle regions of murine plasminogen. A peptide fragment from the same region of human plasminogen (i.e. containing kringles 1-4) also strongly inhibited proliferation of capillary endothelial cells in vitro and in vivo. The intact plasminogen from which this peptide fragment was derived did not possess as potent an inhibitory effect.
Several angiogenesis inhibitors are currently under development for use in treating angiogenic diseases (Gasparini, G. and Harris, A. L., J. Clin. Oncol., 13(3): 765-782, (1995)), but there are disadvantages associated with these compounds. Suramin, for example, is a potent angiogenesis inhibitor but causes severe systemic toxicity in humans at doses required for antitumor activity. Compounds such as retinoids, interferons and antiestrogens are safe for human use but have weak antiangiogenic effects. Still other compounds may be difficult or costly to make.
Thus, there is a need for compounds useful in treating angiogenic diseases in mammals. More specifically, there is a need for angiogenesis inhibitors which are safe for therapeutic use and which exhibit selective toxicity with respect to the pathological condition such as by selectively inhibiting the proliferation of endothelial cells while exhibiting no or a low degree of toxicity to normal (ie. non-cancerous) cells. Such compounds should also be easily and cost-effectively made.
In its principle embodiment, the present invention provides a kringle 5 peptide compound represented by the structural formula A-B-C-X-Y (I) or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein A is absent or a nitrogen protecting group; Y is absent or a carboxylic acid protecting group; B is absent or is from 1 to about 197 naturally-occurring amino acid residues corresponding to the sequence from about amino acid position 334 to amino acid position 530 of SEQ ID NO:1; C is R1-R2-R3-R4 wherein R1 is lysyl; R2 is leucyl or arginyl; R3 is tyrosyl, 3-I-tyrosyl or phenylalanyl; R4 is aspartyl; and X is absent or is from 1 to about 12 naturally-occurring amino acid residues corresponding to the sequence from amino acid position 535 to about amino acid position 546 of SEQ ID NO:1 and homologues and analogues thereof.
The present invention also includes a kringle 5 peptide compound represented by the structural formula A-B1-C1-X1-Y (II) or a pharmaceutically acceptable salt, ester or prodrug thereof wherein A is absent or a nitrogen protecting group; Y is absent or a carboxylic acid protecting group; B1 is absent or is from 1 to about 176 naturally-occurring amino acid residues corresponding to the sequence from about amino acid position 334 to amino acid position 513 of SEQ ID NO:1; C1 is the sequence from amino acid position 514 to amino acid position 523 of SEQ ID NO:1; and X1 is absent or is from 1 to about 10 naturally-occurring amino acid residues corresponding to the sequence from amino acid position 524 to amino acid position 533 of SEQ ID NO:1 and homologues and analogues thereof.
The present invention also includes a method for treating a patient in need of antiangiogenesis therapy comprising administering to the patient a compound containing a kringle 5 peptide fragment or kringle 5 fusion protein.
The present invention also includes a composition for treating a patient in need of anti-angiogenesis therapy comprising a compound containing a kringle 5 peptide fragment or kringle 5 fusion protein, kringle 5 antisera, kringle 5 receptor agonists and antagonists and kringle 5 antagonists linked to cytotoxic agents either alone or in combination with a pharmaceutically acceptable excipient and/or optionally sustained release compounds to form a therapeutic composition.
The present invention also includes a composition for the treatment of a disease selected from the group consisting of cancer, arthritis, macular degeneration and diabetic retinopathy comprising a compound containing a kringle 5 peptide fragment or kringle 5 fusion protein.
The present invention also includes a composition comprising an isolated single or double-stranded polynucleotide sequence that encodes a kringle 5 peptide fragment or fusion protein. Such a polynucleotide is preferably a DNA molecule. The present invention also includes a vector containing a DNA sequence encoding a kringle 5 peptide fragment or fusion protein wherein the vector is capable of expressing a kringle 5 peptide fragment or kringle 5 fusion protein when present in a cell and a composition comprising a cell containing a vector wherein the vector contains a DNA sequence encoding a kringle 5 peptide fragment or kringle 5 fusion protein. The present invention further encompasses gene therapy methods whereby DNA sequences encoding a kringle 5 peptide fragment or kringle 5 fusion protein or kringle 5 peptide fragment conjugate are introduced into a patient to modify in vivo kringle 5 levels.
The present invention also includes a method of making a kringle 5 peptide fragment comprising the steps of: (a) exposing mammalian plasminogen to human or porcine elastase at a ratio of about 1:100 to about 1:300 to form a mixture of said plasminogen and said elastase; (b) incubating said mixture and (c) isolating the kringle 5 peptide fragment from said mixture.
The present invention also includes a method of making a kringle 5 peptide fragment comprising the steps of: (a) exposing mammalian plasminogen to human or porcine elastase at an elastase:plasminogen ratio of about 1:100 to about 1:300 to form a mixture of said elastase and said plasminogen; (b) incubating said mixture; and (c) isolating a protein conjugate of a kringle 5 peptide fragment from said mixture; (d) exposing said protein conjugate of the kringle 5 peptide fragment to pepsin at a ratio of about 1:0.2 to form a mixture of said pepsin and said plasminogen and (d) isolating said kringle 5 peptide fragment from said mixture. Alternatively, a kringle 5 peptide fragment or kringle 5 fusion protein can be made by a method comprising the steps of: (a) isolating a polynucleotide which encodes said kringle 5 peptide fragment or kringle 5 fusion protein; (b) cloning the polynucleotide into an expression vector; (c) transforming the vector into a suitable host cell; and growing the host cell under conditions suitable for the expression of the soluble kringle 5 peptide fragment or kringle 5 fusion protein.